Oxidative stress contributes to cell injury in diabetic nephropathy (DN), a major complication of type 1 and type 2 diabetes. Diabetes is characterized by mitochondrial dysfunction which results in decreased activity of the respiratory chain complexes associated with enhanced generation of reactive oxygen species (ROS). Hyperglycemia contributes to mitochondrial dysfunction and oxidative stress. We have strong evidence that high glucose enhances cell hypertrophy and fibronectin expression in mesangial cells through the NAD(P)H- oxidase isoenzyme Nox4. In addition, Nox4-dependent ROS generation is increased within the renal cortex and isolated glomeruli of rats with streptozotocin-induced diabetes. We provide strong evidence that Nox4 is present not only in cell membranes but also in mitochondria. This suggests that in addition to the mitochondrial electron-transport chain (mtETC), Nox oxidases are a source of ROS in diabetes. The central hypothesis of this proposal is that Nox4 is activated by glucose in renal glomerular cells and that an interplay exists between Nox4 and the mtETC that results in hypertrophy and matrix accumulation. Specific aim #1: to investigate the mechanisms by which glucose activates Nox4 and mtETC and the role of these two sources of ROS in the activation of protein kinase C and NFKB resulting in hypertrophy and fibronectin expression in renal cells. Specific aim #2: to explore the role of Nox4- and mtETC-derived ROS and the activation of PKC and NFKB in the structural and functional changes of diabetic nephropathy in two rat models of type 1 and type II diabetes. This project seeks to identify the cellular and subcellular source(s) of ROS and explore mechanisms by which hyperglycemia and oxidants result in kidney injury. This should help identify specific antioxidants to treat progressive diabetic nephropathy. Diabetes and diabetic nephropathy are major causes of morbidity and mortality in the general population. Euglycemia is a difficult goal to achieve. Oxidative stress contributes to diabetic complications; however, the precise sources of oxygen radicals are not completely defined. It is our hope that identifying specific sources of oxygen radicals will allow targeted therapy to prevent diabetic nephropathy.